Aim I) Modeling human disease genetic heterogeneity in Drosophila. The requirement for >=-secretase in Notch intramembrane proteolysis, together with the wealth of Notch-related molecular and genetic signal outputs that can be analyzed in Drosophila, prompted us to explore the use of transgenic flies to model disease heterogeneity associated with a large set of mutations in the human Presenilin-1 gene. Taking advantage of the relative ease and affordability of producing transgenic Drosophila, we created a collection of 200 transgenic stocks representing 14 mutant variants of Presenilin having different age of onset values in human pedigrees. Analysis of this collection yielded insights into the conserved proteolytic activity of >=-secretase towards different substrates, and established the validity of using Drosophila to study allele-specific features of pathogenic human mutations. Aim II) Endosomal biogenesis and Notch activation. We are investigating the function of a classical mutant termed big brain (bib) in Notch proteolysis and intracellular trafficking. The Bib protein belongs to the aquaporin channel family, which transport water, ions, or small solutes across biological membranes. Our analysis of Bib revealed that it plays an important role in post-endocytic maturation of early endosomes, and causes a failure in Notch signal propagation at a specific membrane trafficking step downstream of >=-secretase-mediated Notch proteolysis. Further studies on the biochemical function of Bib and its relationship to other endosomal factors are currently underway. Aim III) Forward genetic screen for secretory and endosomal trafficking mutants. Several genetic modulators of Notch signaling, including ones characterized previously by our laboratory, were ultimately found to affect the trafficking of Notch and/or its ligands in the secretory and endocytic compartments. These findings prompted us to initiate forward genetic screens to search directly for genes affecting the cell biological distribution of Notch in the highly polarized epithelial cells of the imaginal discs, which continuously require active Notch signaling. We have now identified a large yet relatively specific group of mutant genes that display particular defects in intracellular Notch trafficking. Evaluation and further analysis of these loci are ongoing.